The invention relates generally to ion cyclotron resonance trap mass spectrometry and, more particularly, to the irradiation of ions in an ion cyclotron resonance trap.
Due to its very high mass accuracy and mass resolution, Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) has a unique place in ion-trap mass spectrometry. FT-ICR-MS uses electromagnetic ion traps. In a magnetic field, all ions with a component of motion perpendicular to the magnetic field lines are forced by the Lorentz force to describe cyclotron orbits. Without absorbing additional energy, they are unable to escape in the plane perpendicular to the magnetic field. However, a motion of ions along the magnetic field lines does not cause a Lorentz force, thus, the ions must be trapped in this dimension with an additional electric field. The ion detection is made here by determination of cyclotron frequencies of ions based on image currents in the trap. Since those frequencies are inversely proportional to the m/z ratio (mass divided by the number of charges) of the circling ions, the frequency determination means the determination of the m/z ratio. Nowadays, in analytical FT-ICR-MS generally strong superconducting magnets are used. The FT-ICR mass spectrometry has been reviewed by Marshall, A. G.; Hendrickson, C. L.; Jackson, G. S. xe2x80x9cFourier Transform Ion Cyclotron Resonance Mass Spectrometry: A primerxe2x80x9d Mass Spectrom. Rev. 1998, 17, 1-35.
Electron capture dissociation (ECD) is a relatively new method to fragment ions and to obtain insight into the ion structures using the information from fragment ion spectra. During the ECD process multiply charged ions in an ICR trap capture low energy electrons to produce cationic dissociation products. Multiply charged ions can be generated for example by electrospray ionization. The electron capture dissociation of peptide or protein ions mainly results in c and z type fragment ions. These c and z fragments, which are usually not formed in collision induced dissociation processes (CID, see below), are produced by cleaving the bond between the amino-nitrogen atom, which is involved in the peptide bond, and the neighboring carbon atom from which the amino group originates. The c and z fragments from ECD provide sets of information complementary to those from fragmentation by other ion-fragmentation methods and lead to a more complete determination of the sequences of polypeptides and proteins. The following literature is recommended about the fundamentals and applications of the ECD method: McLafferty, F. W.; Horn, D. M.; Breuker, K.; Ge, Y.; Lewis, M. A.; Cerda, B.; Zubarev, R. A.; Carpenter, B. K. xe2x80x9cElectron Capture Dissociation of Gaseous Multiply Charged Ions by Fourier Transform Ion Cyclotron Resonancexe2x80x9d J. Am. Soc. Mass Spectrom. 2001, 12, 245-249. Zubarev, R. A.; Horn, D. M.; Fridriksson, E. K.; Kelleher, N. L.; Kruger N. A.; Lewis, M. A.; Carpenter, B. K.; McLafferty, F. W. xe2x80x9cElectron Capture Dissociation for Structural Characterization of Multiply Charged Protein Cationsxe2x80x9d Anal. Chem. 2000, 72, 563-573.
Dissociation processes resulting from electron-ion interactions are not limited to ECD. For characterization of negatively charged ions, the electron detachment dissociation (EDD) is used. In the EDD process, an electron is removed from multiply charged ions, while anionic dissociation products are formed (Budnik, B. A.; Haselman, K. F.; Zubarev, R. A. xe2x80x9cElectron Detachment Dissociation of Peptide Di-Anions: An Electron-Hole Recombination Phenomenonxe2x80x9d Chem. Phys. Lett. 2001, 342, 299-302).
The efficiency and rate of ECD is dependent on the electron flux density. The efficiency and rate of ECD can be improved by maximizing the overlap of ions with the electron beam. In conventional FT-ICR mass spectrometry, electrons are generated by a filament, which is placed outside the ICR trap. In most cases, the filament is in the vicinity of the trap and still in the room-temperature bore of the superconducting magnet. Electrons are guided parallel to the magnetic field (axially) into the trap. Due to reasons of thermal conductivity, only a central region of a filament reaches a suitably high temperature and emits electrons. Therefore, the electron beam is usually very thin like a thread in the magnetic field. After the electron beam is formed, attempts to expand this thin beam fail, since every movement perpendicular to the magnetic field produces a Lorenz force at right angles to it, which drives the electrons into tiny cyclotron trajectories. The electron beam must therefore be generated as a wider beam initially. Recently, large-surface electron emitters have been used to produce electrons for the ECD experiments. This way, the electron emitting area has been dramatically increased and the probability of ion-electron interactions in the ICR trap leading to dissociation is increased. In fact, by using these new emitters, improved ECD results have been obtained (Tsybin, Y. O.; Hakansson, P.; Budnik, B. A.; Haselmann, K. F.; Kjeldsen, F.; Gorshkov, M.; Zubarev, R. A.; xe2x80x9cImproved Low Energy Electron Injection Systems for High Rate Electron Capture Dissociation in Fourier Transform Ion Cyclotron Resonance Mass Spectrometryxe2x80x9d Rapid Commun. Mass Spectrom. 2001, 15, 1840-1854; as well as the world patent application WO 02/078048 A1 published in October 2002.).
In FT-ICR MS, one would like to study the interaction of stored ions with photons as well. It is possible to irradiate and excite ions with photons. As a result of this excitation ions can be fragmented too (photodissociation). The photons can be generated from ultraviolet, visible or infrared light, which may also be a laser beam.
A photo-induced fragmentation method which is increasingly used in FT-ICR mass spectrometry is infrared multiphoton dissociation (IRMPD). In this case, the ions are excited by multiple, sequentially absorbed infrared photons produced by an infrared laser (e.g. a CO2 laser). Subsequently, a dissociation process is observed which produces similar results to the widely used collision induced dissociation (CID) method. For mass spectrometric methods such as FT-ICR, which require a very good ultra-high vacuum, IRMPD is a popular alternative since, in this case, no collision gas has to be xe2x80x9cpulsed inxe2x80x9d to fragment the ions. Similar to the CID, the so-called b- and y-type fragment ions from peptide or protein ions are also produced in IRMPD experiments. These ions are obtained by a cleavage of the bond between the peptide nitrogen atom and the (neighboring) carboxyl carbon atom. IRMPD is not only used in sequencing polypeptides and proteins, it is also generally used to investigate the higher order structures of biomolecules and their dynamics. When using the infrared laser to obtain a dissociation spectrum which will allow an identification of a substance, the irradiation time is generally less than 500 ms in FT-ICR mass spectrometry. In order to induce an infrared multiphoton dissociation, the IR laser beam must be introduced into a region where the ions are present. The interaction of ions with the laser beam can best be studied in an ion trap (Paul trap, Penning trap, ion cyclotron trap or linear RF multipole trap). For infrared multiphoton dissociation experiments in one of these traps, an infrared laser beam is introduced, usually axially to the trap and in most cases through the aperture of one of the end plates (end plates in the linear multipole trap, trapping plates in the FT-ICR trap or end caps in the Paul trap). Examples of literature which deals with the use of IRMPD applications are: Little, D. P.; Speir, J. P.; Senko, M. W.; O""Connor, P. B.; McLafferty, F. W. xe2x80x9cInfrared Multiphoton Dissociation of Large Multiply-charged Ions for Biomolecule Sequencingxe2x80x9d Anal. Chem. 1994, 66, 2809-2815; Colorado, A.; Shen, J. X.; Vartanian, V. H.; Brodbelt J. xe2x80x9cUse of Infrared Multiphoton Photodissociation with SWIFT for Electrospray Ionization and Laser Desorption Applications in a Quadrupole Ion Trap Mass Spectrometerxe2x80x9d Anal Chem. 1996, 68, 4033-4043; Hofstadler, S. A.; Sannes-Lowery, K. A.; Griffey R. A. xe2x80x9cInfrared Multiphoton Dissociation in an External Ion Reservoirxe2x80x9d Anal. Chem. 1000, 71, 2067-2070.
With the introduction of the new ECD fragmentation method, methods for introducing electrons into the ICR trap became especially popular. ECD experiments have to be carried out with low energy electrons. One of the axial entrances of the ICR trap is normally used for the introduction of ions (which are generated in an external ion source) into the trap. The other axial entrance is often used for infrared multiphoton dissociation experiments.
Since only shielded ICR magnets are used nowadays, the use of an external, non-axial electron source, of which the electrons would follow the magnetic field lines to enter the ICR trap, is complicated. With shielded superconducting magnets, the magnetic field shows (axially) a dramatic gradient only in the immediate vicinity of the geometric limits of the magnet housing. In order to achieve efficient electron injection into the ICR trap, the electron source is positioned in a region where there is a high (and uniform) magnetic field. At locations where a non-axial electron source is usually placed outside the magnet, the fringing fields of a shielded magnet is not strong enough. Since the external ion sources (external to the ion trap) are constantly used in FT-ICR mass spectrometry and IRMPD is very often used (laser beam enters from the other side of the ICR trap), it is almost impossible to additionally install an electron source axial to the trap.
FIG. 1 (prior art) shows a Fourier transform ion cyclotron resonance mass spectrometer with an external ion source (1). The ion source (1) in the figure is only shown schematically. Ions (2) which are generated in this source are transferred to the ICR trap (4) using a special ion transfer lens system (3). The figure shows a cylindrical ICR trap as an example. The ion beam (5) is guided into the ion trap axially from the source end through the aperture (6) in the trapping plate (7) on the left. The ICR trap is located in a magnetic field (aligned, for example, in the direction of the arrow 8) co-axially to the field produced by a superconducting magnet (9). Nowadays, magnetic fields of magnetic inductions from 3 to 12 Tesla are used in commercial FT-ICR systems. Ions are captured and stored in the ICR trap and excited and detected later on.
A typical experiment using stored ions is the infrared multiphoton dissociation experiment. An infrared laser (for example a CO2 laser) (10), mounted behind the magnet, projects its beam into the ICR trap through a laser window (11) and through the aperture of the trapping plate on the right (12). In this specific set up, the laser beam (13) is deflected by a mirror (14) by 90xc2x0. Ions in the ICR trap get excited by sequentially absorbing a number of the IR photons, and dissociate.
The formation of the fragment ions generated by the infrared multiphoton dissociation (IRMPD) is closely correlated to the structure of the initial ion and its chemical bonding conditions. This method is therefore used in mass spectrometry for ion structure determination. Some details shown in the figure are the vacuum stage partitions (15) and (16) and the three pump nozzles (17), (18) and (19). This figure clearly shows that the two axial introductions into the ion cyclotron resonance trap are occupied. The ions generated outside the trap are introduced into the trap on one side and the infrared laser beam is introduced on the other side. Since one side (left side in FIG. 1) is almost always occupied with the ion introduction, the other side (right side in FIG. 1) has to be used for introducing beams or particles. According to the prior art, a constant switching between the laser window and electron sources, etc., is necessary. This process requires a venting of the ultrahigh vacuum. Therefore, the prior art excludes the simultaneous application of experimental methods that require the introduction of photons as well as electrons into the ICR trap. Neither does the prior art allow sequential use of these techniques on the very same batch (ensemble) of stored ions (in the ICR trap).
The use of sliding and rotating feedthroughs for moving the ion and electron sources solves the problem to a very limited extent, as these methods are generally awkward and slow. In the ultra-high vacuum system used in Fourier transform mass spectrometry in the range of 10xe2x88x9210 mbar, placing a sliding or rotating feedthrough with the corresponding vacuum locks is an option that requires intensive work.
For experiments which require simultaneous irradiation of ions with photons and low energy electrons, these slow and time consuming methods of switching from electron beam to photon beam (and back) are basically useless. Sequential ECD and IRMPD experiments for kinetic analysis of the assembly of ions stored in the ICR trap require faster switching methods. Recently, studies of protein ions in the ICR trap using ECD and IRMPD have indicated that protein ions undergo rapid structural changes (e.g. folding and unfolding) (Hom, D. M.; Breuker, K.; Frank, A. J.; McLafferty, F. W. xe2x80x9cKinetic Intermediates in the Folding of Gaseous Protein Ions Characterized by Electron Capture Dissociation Mass Spectrometryxe2x80x9d J. Am. Chem. Soc. 2001, 123, 9792-9799).
Since electron capture dissociation provides important complementary results for infrared multiphoton dissociation, it is advantageous for users of FT-ICR mass spectrometers to apply ECD and IRMPD on the test substances simultaneously. Thus, it is important to be able to switch between the fragmentation methods without time consuming mechanical operations. It is also desirable to be able to use ECD and IRMPD on the same group of ions, if possible, in the same experimental sequence. However, since filaments or larger cathodes (such as dispenser cathodes or other indirectly heated cathodes) are used for electron capture dissociation, these block the path into the ICR trap and, for example, a laser beam can no longer be introduced at the same time.
The idea of the invention is to be able to use a hollow (or tubular) electron beam and light beams simultaneously. The tubular electron beam is provided by an electron injection system that contains a hollow electron emitter which has a bore in it. The electron injection system therefore enables the passage of light beams parallel to the magnetic field in which the ICR trap is located. The light beams (also laser light) can be in the ultraviolet (UV), visible (VIS) or infrared (IR) range.
A ring shaped (hollow cylindrical) electron emitter (hollow cathode, ring cathode) placed at one end of the ICR trap produces an annular electron cloud which can be extracted along the magnetic field lines in the direction of the ICR trap. By continuous or pulsed extraction of this electron cloud, a hollow tubular electron beam is produced. It is also possible to control the flux of the electrons in the tubular electron beam by using suitable devices, such as grids, apertured diaphragms and ring shaped electrodes. This way, multiply charged ions can dissociate in the ICR trap by capturing low energy electrons (electron capture dissociation). Low energy electrons are defined as electrons with kinetic energies Ekxe2x89xa630 eV and, in particular, Ekxe2x89xa61 eV.
An infrared laser beam (usually non-focused and about 2 mm wide) which enters the ICR trap is needed for the IRMPD experiments in the ICR trap. Since the present invention uses a hollow electron emitter (hollow cathode, ring cathode) for generating electrons, the aperture in the emitter enables the infrared beam to be introduced even while the hollow cathode is in operation. This combination makes it possible to operate the electron emitter alone in order to study electron-ion interactions (such as electron capture dissociation) or to switch on the light beam and study ion-photon interactions (such as the photodissociation of ions). This invention also allows simultaneous irradiation with electrons and photons. Of course, it is possible to ionize neutral molecules in the ICR trap both with electrons and with photons.
Positive ions which are located in the ICR cell can be further ionized by interacting with an electron beamxe2x80x94in a similar way to electron-impact ionizationxe2x80x94where a change in the charge number happens. For this purpose, however, electrons with a higher kinetic energy are necessary.
A further use for the invention is electron detachment dissociation (EDD) of multiply negatively charged ions in the ICR trap. In this case, the interaction of the ion of interest with an electron leads to the detachment of an electron and dissociation of the ion.